Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes, such as cytokinesis. Recently, this laboratory and another laboratory (Katsuragawa et al., Eur. J. Biochem. 184: 611-616, 1989) have isolated two different cDNAs which encode two different chicken nonmuscle myosin heavy chains (MHCs). In order to understand the biological significance of MHC isoforms in nonmuscle cells, we studied the expression of two types of MHCs in a number of chicken tissues at different stages of development and quantitated the relative contents of mRNA encoding the MHC isoforms in a number of tissues, using RNA blot analysis with two specific oligonucleotide probes. Our results show that the relative content of mRNA encoding MHC-A and MHC-B differs in a tissue-dependent manner. The relative content of mRNA encoding MHC-A vs MHC-B varies from greater than 9:1 in spleen and intestinal epithelial cells, to 6:4 in kidney and 2:8 in brain. Using SDS-polyacrylamide gels, we have separated two nonmuscle MHC isoforms (196 and 198 kD) which can be distinguished from each other and from the gizzard smooth muscle MHCs by peptide mapping. Spleen and intestinal epithelial cells contain almost exclusively 196 kD MHC, whereas brain contains predominantly 198 kD MHC. Kidney contains both 196 and 198 kD MHCs. These results suggest that MHC-A mRNA encodes the 196 kD polypeptide and MHCB mRNA encodes the 198 kD polypeptide. We also studied the effect of serum on MHC mRNA expression in cultured chicken embryo fibroblasts as well as on primary cultures of aorta smooth muscle cells. Serum stimulation results in a three-fold increase in the mRNA encoding MHCA and a three-fold decrease in mRNA encoding MHC-B at 6 h. The maximum changes in both MHC-A and B mRNA expression occur during G1 phase. Actinomycin D, an inhibitor of transcription, abolished the serum-induced changes in both MHC-A and B mRNAs, suggesting that these changes are regulated at the transcriptional level.